Many, and possibly all, human cancers contain mutations that lead to the loss of the wild-type retinoblastoma tumor suppressor protein (pRB) or to the untimely phosphorylation, and hence functional inactivation, of pRB by cyclin dependent kinases (cdk). How cdks recognize substrates in general, and pRB in particular, is poorly understood and will form the basis of specific aim 1. In this regard, they recently identified a short, colinear, motif which will target cyclin A/cdk2 complexes to potential substrates such as E2F1 and p107. Their preliminary biochemical data suggest that the C-terminus of pRB contains a sequence, or sequences, which, at least at the level of secondary structure, resembles this motif. The ability of pRB to regulate cell growth in vitro and to suppress tumor formation in vivo appears to be due, at least in part, to its ability to form pRB/E2F complexes which actively repress transcription from E2F responsive promoters. Using a panel of pRB mutants, we have recently found that the ability of pRB to bind to E2F can be dissociated from its ability to cooperate with certain non-E2F transcription factors to promote differentiation. Two naturally occurring, partially penetrant, pRB mutants were unable to bind to E2F but were wild-type for the ability to promote differentiation in vitro In contrast, classical null pRB mutants were defective for both of these functions. The lower risk of retinoblastoma associated with the partially penetrant alleles (~1/10 of a null allele) suggests that this differentiation promotion of pRB contributes to tumor suppression in vivo. Testing this hypothesis in a mouse model, as well as determining the biochemical basis for this activity, will form the basis of specific aim 2.